1. Field of the Invention
The invention relates to compositions useful in the treatment and prevention of blood disorders such as anemia, thalassemia and sickle cell disease. Compositions comprise proteins or chemicals that stimulate the specific expression of a globin protein or the proliferation or development of hemoglobin expressing or other myeloid cells. The invention also relates to methods and medical aids which utilize these compositions to ameliorate symptoms associated with blood disorders.
2. Description of the Background
Hematopoiesis, or the formation of blood cells, begins in the developing human embryo as clusters of stem cells called blood islands. These cells appear in the yolk sac at about the third week of development and, at about the third month, migrate to the developing liver which becomes the principal site of blood cell formation. Although the spleen, lymph nodes and bone marrow all make small contributions to blood cell development, not until the fourth month does the bone marrow become the principal site of hematopoiesis. At birth, virtually all blood cells originate from the bone marrow. Although small foci of blood-forming cells sometimes persist in the liver for longer periods of time, hepatic blood cell formation has decreased to a trickle. At this time, all of the marrow is actively forming blood cells and continues to do so until after puberty when, at about 18 years of age, the principal sites of blood cell formation become the marrow of the vertebrae, ribs, sternum, skull, pelvis and the proximal epiphyseal regions of the femur and humerus. These areas represent only about half of the available marrow. The cavities which remain are filled with yellow-fatty tissues.
In the adult, hematopoiesis involves the bone marrow, the lymph nodes and the spleen. These organs and associated tissues are traditionally divided into myeloid and lymphoid tissue-types. Myeloid tissues and the cells derived from the myeloid tissue include the erythrocytes, platelets, granulocytes and monocytes. Lymphoid and lymphoid-derived tissues include the thymus, lymph nodes and spleen. The myeloid/lymphoid division is somewhat artificial as these two types of tissues are believed to originate from a single pluripotent stem cell.
Lymphoid and myeloid stem cells, formed from division of the pluripotent cell, are precursors for all subsequent cell types (FIG. 1). The committed cell-types for the lymphoid stem cell include the pro-T cells which form mature T cells and the pro-B cells which differentiate into plasma cells. Intermediate cell types can be distinguished based on cell-surface phenomenon such as the expression of immunoglobulin heavy and light chain, Ia protein and other cell surface markers. The three committed cell-types for the myeloid stem cell include E/mega cells which differentiate into the erythrocyte-burst forming unit (BFU-E) followed by the erythrocyte-colony forming unit cells (CFU-E) and megakaryocyte-CFU cells (CFU-mega), granulocyte/macrophage-CFU cells (CFU-G/M) which differentiate into CFU-G and CFU-M cells, and the eosinophil-CFU cells (CFU-Eo) which ultimately form mature eosinophils. Although these committed cell types reside mainly in the marrow, some circulate throughout the body in the blood stream.
The bone marrow provides a unique environment for pluripotent and committed cells. It contains both structural and humoral components that have yet to be successfully duplicated in culture. The marrow cavity itself is a network of thin-walled sinusoids lined with endothelial cells. Between the walls of bone are clusters of hematopoietic cells and fat cells constantly fed by mature blood cells entering through the endothelium. Differentiated cells ready to function within the circulatory system depart the cavity in a similar fashion.
The relative proportions of cell types in the bone marrow have a myeloid/erythroid ratio of about three to one comprising about 60% granulocytes and their precursors, about 10% lymphocytes and their precursors, about 20% erythrocytes and their precursors, and about 10% unidentified cells. The predominant myeloid cell types in the marrow cavity are the myelocytes, metamyelocytes and granulocytes. The predominant cell types in the erythroid compartment are the polychromatophilic and orhtochromic normoblasts. Under conditions of normal iron metabolism, about 30% to 40% of the normoblasts contain scattered ferritin granules. These cells are referred to as sideroblasts and the iron granules they contain are reservoirs drawn from as the cells insert iron into protoporphyrin to form heme. The production of heme and the production of globin are precisely balanced within the cell. If either is hindered or depressed, for whatever reason, excess ferritin accumulates in the sideroblasts. This increased iron accumulation can be visualized in the mitochondria, the loci of heme synthesis.
The major function of red blood cells is to transport oxygen to tissues of the body. Minor functions include the transportation of nutrients, intercellular messages and cytokines, and the absorption of cellular metabolites. Anemia, or a loss of red blood cells or red blood cell capacity, can be grossly defined as a reduction in the ability of blood to transport oxygen and may be acute or chronic. Chronic blood loss may be caused by extrinsic red blood cell abnormalities, intrinsic abnormalities or impaired production of red blood cells. Extrinsic or extra-corpuscular abnormalities include antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical trauma to red cells such as micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation. In addition, infections by parasites such as Plasmodium, chemical injuries from, for example, lead poisoning, and sequestration in the mononuclear system such as by hypersplenism can provoke red blood cell disorders.
Impaired red blood cell production can occur by disturbing the proliferation and differentiation of the stem cells or committed cells. Some of the more common diseases of red cell production include aplastic anemia, hypoplastic anemia, pure red cell aplasia and anemia associated with renal failure or endocrine disorders. Disturbances of the proliferation and differentiation of erythroblasts include defects in DNA synthesis such as impaired utilization of vitamin B.sub.12 or folic acid and the megaloblastic anemias, defects in heme or globin synthesis, and anemias of unknown origins such as sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HIV, hepatitis virus or other viruses, and myelophthisic anemias caused by marrow deficiencies.
Intrinsic abnormalities include both hereditary and acquired disorders. Acquired disorders are those which have been induced through, for example, a membrane defect such as paroxysmal nocturnal hemoglobinuria. Hereditary disorders include disorders of membrane cytoskeleton such as spherocytosis and elliptocytosis, disorders of lipid synthesis such as an abnormally increased lecithin content of the cellular membrane, red cell enzyme deficiencies such as deficiencies of pyruvate kinase, hexokinase, glutathione synthetase and glucose-6-phosphate dehydrogenase. Although red blood cell disorders may be caused by certain drugs and immune system disorders, the majority are caused by genetic defects in the expression of hemoglobin. Disorders of hemoglobin synthesis include deficiencies of globin synthesis such as thalassemia syndromes and structural abnormalities of globin such as sickle cell syndromes and syndromes associated with unstable hemoglobins.
Hemoglobin comprises four protein chains, two alpha chains and two beta chains (.alpha..sub.2 .beta..sub.2), interwoven together, each with its own molecule of iron and with a combined molecular weight of about 68 kD. The hemoglobin macromolecule is normally glycosylated and upon absorbing oxygen from the lungs transforms into oxyhemoglobin (HbO.sub.2). There are at least six distinct forms of hemoglobin, each expressed at various times during development. Hemoglobin in the embryo is found in at least three forms, Hb-Gower 1 (.xi..sub.2 .epsilon..sub.2), Hb-Gower 2 (.alpha..sub.2 .epsilon..sub.2), and Hb-Portand (.xi..sub.2 .gamma..sub.2). Hemoglobin in the fetus comprises nearly totally HbF (.alpha..sub.2 .gamma..sub.2), whereas hemoglobin in the adult contains about 96% HbA (.alpha..sub.2 .beta..sub.2), about 3% HbA.sub.2 (.alpha..sub.2 .delta..sub.2) and about 1% fetal HbF (.alpha..sub.2 .gamma..sub.2). The embryonic switch of globin expression from .xi. to .alpha. and from .epsilon. to .gamma. begins in the yolk sac. However, chains of embryonic .xi. and .epsilon. have been found in the fetal liver and complete transition to the fetal form does not occur until late in fetal development. The fetal switch from .gamma. to .beta. begins later in erythropoiesis with the amount of .gamma. globin produced increasing throughout gestation. At birth, .beta. globin accounts for about 40% of non-a globin chain synthesis and thereafter continues to rapidly increase. Neither the switch from embryonic to fetal or fetal to adult appears to be controlled through cell surface or known cytokine interactions. Control seems to reside in a developmental clock with the switch occurring at times determined only by the stage of fetal development.
Defects or mutations in globin chain expression are common. Some of these genetic mutations pose no adverse or only minor consequences to the person, however, most mutations prevent the formation of an intact or normal hemoglobin molecule through a functional or structural inability to effectively bind iron, an inability of the chains or chain pairs to effectively or properly interact, an inability of the molecule to absorb or release oxygen, a failure to express sufficient quantities of one or more globin chains or a combination of these malfunctions. For example, substitutions of valine for glutamic acid at the sixth position of the .beta. chain produces HbS and was found to occur in about 30% of black Americans. In the HbS heterozygote, only about 40% of total hemoglobin is HbS with the remainder being the more normal HbA.
Upon deoxygenation, HbS molecules undergo aggregation and polymerization ultimately leading to a morphological distortion of the red cells which acquire a sickle or holly-leaf shape. Sickling has two major consequences, a chronic hemolytic anemia and an occlusion of small blood vessels that results in ischemic damage to tissues. Further, when exposed to low oxygen tensions, polymerization converts HbS hemoglobin from a free-flowing liquid to a viscous gel. Consequently, the degree of pathology associated with sickle cell anemia can be correlated with the relative amount of HbS in the patient's system.
Individuals with severe sickle cell anemia develop no symptoms until about five to six months after birth. In these infants it was determined that fetal hemoglobin did not interact with HbS and, as long as sufficient quantities were present, could modulate the effects of HbS disease. This modulating effect of .beta. globin is also observed with other .beta. globin disorders, such as HbC and HbD, and other mutations of the .beta. chain. HbS polymerization is also significantly affected by the hemoglobin concentration of the cell. The higher the HbS concentration, the greater the chances for contact between two or more HbS molecules. Dehydration increases hemoglobin concentration and greatly facilitates sickling.
To some extent, sickling is a reversible phenomenon. With increased oxygen tensions, sickled cells depolymerize. This process of polymerization-depolymerization is very damaging to red cell membranes and eventually leads to irreversibly sickled cells (ISC) which retain their abnormal shape even when fully oxygenated. The average ISC survives for about 20 days in the body, as compared to the normal 120 day life span. Individuals with HbS syndromes have frequent infections, chronic hemolysis with a striking reticulocytosis and hyperbilirubinemia. The course of the disease is typically punctuated with a variety of painful crises called vaso-occlusive crises. These crises represent episodes of hypoxic injury and infarction in the organs, abdomen, chest, extremities or joints. Leg ulcers are an additional manifestation of the vaso-occlusive tendency of this disease. Central nervous system involvement is common producing seizures and even strokes. Aplastic crises, also common, represent a temporary cessation of bone marrow activity and may be triggered by infections, folic acid deficiency or both. Crises are episodic and reversible, but may be fatal. Damage from crisis episodes tends to be cumulative and even in those individuals with milder forms of sickle cell disorders, life-spans can be greatly reduced. Absent alternative intervention, patients typically die before the age of 30.
Anti-gelling compounds including clofibric acid (ClC.sub.6 H.sub.5 OC(CH.sub.3).sub.2 COOH), p-chloro phenoxy acetic acid (ClC.sub.6 H.sub.5 OCH.sub.2 COOH), and phenoxy acetic acid (C.sub.6 H.sub.5 OCH.sub.2 COOH) have been shown to prophylactically inhibit polymerization in artificially deoxygenated blood (D. J. Abraham et al., J. Med. Chem. 25:1015-17, 1982). It was speculated that these compounds may be useful in a narrow respect to prevent blood cell sickling in sickle cell disease. Such treatments may potentially decrease the frequency of symptomatic episodes caused by vaso-occlusive crises if enough of the chemical can be administered to bind all hemoglobin in the body.
The thalassemia syndromes are a heterogenous group of disorders all characterized by a lack of or a decreased synthesis of the globin chains of HbA. Deficiencies of .beta.-globin expression are referred to as .beta.-thalassemias and deficiencies of .alpha.-globin, .alpha.-thalassemias. The hemolytic consequences of deficient globin chain synthesis result from decreased synthesis of one chain and also an excess of the complementary chain. Free chains tend to aggregate into insoluble inclusions within erythrocytes causing premature destruction of maturing erythrocytes and their precursors, ineffective erythropoiesis, and the hemolysis of mature red blood cells. The underlying defects of hemoglobin synthesis have been elucidated over the years and largely reside in the nucleic acid sequences which express or control the expression of .alpha. or .beta. globin protein.
Mammalian globin gene expression is highly regulated during development. The basic structure of the .alpha. and .beta. globin genes are similar as are the basic steps in synthesis of .alpha. and .beta. globin. There are at least five human a globin genes located on chromosome 16 including two adult a globin genes of 141 amino acids that encode identical polypeptides which differ only in their 3'-untranslated regions, one embryonic .alpha. gene, zeta (.xi.), and at least two pseudo-alpha genes, psi zeta (.psi..xi.) and omega alpha (.omega..alpha.). Surprisingly, .alpha.-thalassemias tend to be less severe than .beta. thalassemias. Homozygous pairs of .beta. chains are believed to be more soluble than those derived from unpaired .alpha. chains. Consequently, the effects associated with free or improperly paired globin chains, which correlate with at least half of the clinical pathology associated with thalassemia, are minimized.
Hemoglobin H disease, a more severe form of .alpha. thalassemia, is a deletion of three of the four .alpha. globin genes. It is rarely found in those of African origin, but mostly in Asians. With only a single .alpha. gene, .alpha. chain expression is markedly depressed and there is an excess of .beta. chains forming tetramers called HbH hemoglobin. HbH is unable to withstand oxidative stress and precipitates with vessels or is removed by the spleen. The most severe form of .alpha. thalassemia is hydrops fetalis and results from a deletion of all .alpha. globin genes. In the fetus, tetramers of .gamma. globin develop (Hb Barts) that have an extremely high oxygen affinity and are unable to release oxygen to the tissues. Severe tissue anoxia results and leads to intrauterine fetal death.
The human .beta. globin gene cluster includes one embryonic gene, epsilon (.epsilon.), two adult beta globin genes, beta (.beta.) and delta (.delta.), two fetal beta globin genes G-gamma (G-.gamma.) and A-gamma (A-.gamma.), which differ by only one amino acid, and at least one pseudo-beta gene, psi beta (.psi..beta.). All are expressed from a single 43 kilobase segment of human chromosome 11 (E. F. Fritsch et al., Nature 279:598-603, 1979). Fetal .beta.-type globin, or .gamma. globin, is expressed in the earliest stages of mammalian development and persists until about 32 to 34 weeks of gestation. At this stage, the adult forms of .beta. globin begin to be expressed and substitute for the fetal proteins. Studies correlating clinical hematological results with the locations of various mutations that correspond to switching indicate that a region located upstream of the 5'-end of the .delta.-gene may be involved in the cis suppression of .gamma.-gene expression in adults (E. F. Fritsch et al., Nature 279:598-603, 1979). The reason for this switch from fetal to adult protein is unknown and does not appear to provide any significant benefit to the adult.
Each .beta. globin gene comprises three exons which encode about 146 amino acids, two introns and a 5'-untranslated region containing the promoter sequences. Biosynthesis of .beta. globin begins with transcription of the entire gene followed with RNA processing of the message, removal of the introns by splicing, poly A addition, capping and post-transcriptional modifications. The mature mRNA molecule is exported from the nucleus and translated into .beta. globin. Defects in each of these functions have been found associated with specific thalassemias. Identified mutations include single-nucleotide deletions, insertions and substitutions, frame shift mutations, deletions of entire segments of coding or controlling regions, improper termination signals, aberrant splicing signals, and multiple mutations. .beta..degree.-thalassemias are characterized by a complete absence of any .beta. globin chains. .beta..sup.+ -thalassemias are characterized by a detectable presence of a reduced amount of .beta. chains.
There are three principal categories of .beta.-thalassemia, thalassemia major, thalassemia intermedia and thalassemia minor. Patients with thalassemia minor may be totally asymptomatic and are genotypically .beta..sup.+ /.beta. or .beta..degree./.beta.. Although red cell abnormalities can be detected, symptoms are mild. Thalassemia intermedia patients are most often genotypically .beta..sup.+ /.beta..sup.+ or .beta..degree./.beta. and present severe symptoms which can be alleviated with infrequent blood transfusions. In contrast, thalassemia major patients are genotypically .beta..degree./.beta..degree., .beta..degree./.beta..sup.+ or .beta..sup.+ /.beta..sup.+, and require regular and frequent transfusions. Children suffer from severe growth retardation and die at an early age from the profound effects of anemia. Those that survive longer suffer from morphological changes. The face becomes distorted due to expansion of marrow within the bones of the skull, hepatosplenomegaly ensues, there is a delayed development of the endocrine organs including the sexual organs, and a progressive iron overload with secondary hemochromatosis.
There are two direct consequences of .beta.-thalassemia. First, there is an inadequate formation of HbA and, therefore, an impaired ability to transport oxygen. There are also multiple effects attributable to an imbalance between .alpha. and .beta. chain synthesis. Surprisingly, the pathological consequences of globin chain imbalance appears to be the mote severe. Free .alpha. chains form unstable aggregates that precipitate within red cell precursors in the form of insoluble inclusions. These inclusions damage cellular membranes resulting in a loss of potassium. The cumulative effect of these inclusions on the red blood cells is an ineffective erythropoiesis. An estimated 70% to 85% of normoblasts in the marrow are eventually destroyed. Those that do escape immediate destruction are at increased risk of elimination by the spleen where macrophages remove abnormal cells. Further, hemolysis triggers an increased expression of erythropoietin which expands populations of erythroid precursors within bone marrow and leads to skeletal abnormalities. Another severe complication of .beta. thalassemia is that patients tend to have an increased ability to absorb dietary iron. As most treatments for thalassemia involve multiple transfusions of red blood cells, patients often have a severe state of iron overload damaging all of the organs and particularly the liver. To reduce the amount of iron in their systems, iron chelators are typically administered. Although helpful, patients succumb at an average of between about 17 to 35 years of age to the cumulative effects of the disease and iron overload.
Genotypic variation in healthy individuals have been identified- wherein adult .beta. globin is not formed, but severe complications are avoided. These patients constituitively express fetal or .gamma. globin protein in amounts sufficient to substitute for the missing .beta. globin protein. This hereditary persistence of fetal hemoglobin (HPFH) may involve one or both of the fetal .beta.-globin genes, A-.gamma. and G-.gamma.. Apparently, consistent production of either .gamma.-globin protein accomplishes the necessary functions, at least in the short term, of the abnormal or missing .beta.-globin protein (R. Bernards et al., Nuc. Acids Res. 8:1521-34, 1980).
A variety of small molecules have been shown to effect hemoglobin or fetal globin expression. Early experiments demonstrated that acetate (CH.sub.3 COOH), propionate (CH.sub.3 CH.sub.2 COOH), butyrate (CH.sub.3 CH.sub.2 CH.sub.2 COOH) and isobutyrate (CH.sub.3 CH(CH.sub.3)COOH) all induced hemoglobin synthesis in cultured Friend leukemia cells (E. Takahashi et al., Gann 66:577-80, 1977). Additional studies showed that polar compounds, such as acid amides, and fatty acids could stimulate the expression of both fetal and adult globin genes in murine erythroleukemia cells (U. Nudel et al., Proc. Natl. Acad. Sci. USA 74:1100-4, 1977). Hydroxyurea (H.sub.2 NCONHOH), another relatively small molecule, was found to stimulate globin expression (N. L. Ietvin et al., N. Engl. J. Med. 310:869-73, 1984). Stimulation, however, did not appear to be very specific to fetal globin (S. Charache et al., Blood 69:109-16, 1987). Hydroxyurea is also a well-known carcinogen making its widespread and long term use as a pharmaceutical impractical.
Expression from the .gamma.-globin genes has been successfully manipulated in vivo and in vitro using agents such as cytosine arabinoside (AraC), a cytotoxic agent that induces fetal reticulocyte production (P. Constantoulakis et al., Blood 74:1963-71, 1989), and 5-azacytidine (AZA), a well-known DNA methylase inhibitor (T. J. Ley et al., N. Engl. J. Med. 307:1469-75, 1982). Continuous intravenous administration of AZA produced a five- to seven-fold increase in .gamma. globin mRNA of bone marrow cells (T. J. Ley et al., Blood 62:370-380, 1983). Additional studies have shown that there are significant alterations in the population of stem cells in the bone marrow after AZA treatment (A. T. Torrealba-De Ron et al., Blood 63:201-10, 1984). These experiments indicate that AZA's effects may be more attributable to reprogramming and recruitment of erythroid progenitor cells than to any direct effects on specific gene expression. Many of these agents including AZA, AraC and hydroxyurea are myelotoxic, carcinogenic or teratogenic making long-term use impractical.
One of the major breakthroughs in the treatment of hemoglobinopathies was made when it was discovered that butyric acid (butanoic acid; CH.sub.3 CH.sub.2 CH.sub.2 COOH) accurately and specifically stimulated transcription of the human fetal (.gamma.) globin gene (G. A. Partington et al., EMBO J. 3:2787-92, 1984). These findings were quickly confirmed in vivo wherein it was shown that pharmacological doses of butyric acid greatly increased expression of fetal globin in adult chickens rendered anemic by injections with phenylhydrazine (G. D. Ginder et al., Proc. Natl. Acad. Sci. USA 81:3954-58, 1984). Selective transcriptional activation was again thought to be due to hypo-methylation of the embryonic gene (L. J. Burns et al., Blood 72:1536-42, 1988). Others speculated that histone acetylation, a known effect of butyric acid, may be at least partly responsible for increased fetal gene expression (L. J. Burns et al., EMBO J. 3:2787, 1984).
Over 50 derivatives of butyric acid have since been found to be effective in stimulating fetal globin production (S. P. Perrine et al., Biochem. Biophys. Res. Commun. 148:694-700, 1987). Some of these include butyric acid salts such as sodium and arginine butyrate, .alpha.-amino-n-butyric acid (butyramide; CH.sub.3 CH.sub.2 CH.sub.2 CONH.sub.2), and isobutyramide (CH.sub.3 CH(CH.sub.3)CONH.sub.2). Although promising in pilot clinical studies, treated patients were unable to maintain adequate levels of fetal globin in their system. It was later determined that many of these forms of butyric acid had extremely short-half lives. Oxidation in the serum, clearance by hepatocytes and filtration through the kidneys rapidly eliminated these agents from the patient's system. With others, patients rapidly developed tolerance or metabolites of compounds had the opposite desired effect.
Recently, a number of aliphatic carboxylic acids were tested for their ability to specifically increase fetal globin expression in K562 human erythroleukemia cells (S. Safaya et al., Blood 84:3929-35, 1994). Although longer chains were considered toxic to cells, propionate (CH.sub.3 CH.sub.2 COOH) and valerate (pentanoic acid; CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2 COOH) were found to be most effective. Butyrate (CH.sub.3 (CH.sub.2).sub.2 COOH), caproate (CH.sub.3 (CH.sub.2).sub.4 COOH), caprylate (CH.sub.3 (CH.sub.2).sub.6 COOH), nonanoate (CH.sub.3 (CH.sub.2).sub.7 COOH), and caprate (CH.sub.3 (CH.sub.2).sub.6 COOH) produced much less of an effect. Phenyl acetate (C.sub.6 H.sub.5 CH.sub.2 COOH) and its precursor, 4-phenyl butyrate (C.sub.6 H.sub.5 CH.sub.2 CH.sub.2 CH.sub.2 COOH), were found to decrease fetal globin expressing reticulocyte proliferation, but increase relative proportions of fetal globin per cell in cultured erythroid progenitor cells (E. Fibach et al., Blood 82:2203-9, 1993). Acetate (CH.sub.3 COOH), a metabolic product of butyrate catabolism, increased both erythrocyte precursor populations and also fetal globin synthesis. However, these studies also demonstrated that positive effects could only be maintained for very short periods of time (B. Pace et al., Blood 84:3198-204, 1994).
Other methodologies to increase fetal globin expression have focused on recruitment and reprogramming of erythroid progenitor cells to express fetal globin Agents tested in vivo or in vitro using this approach include hematopoietic growth factors such as erythropoietin (EPO) (Al-Khatti et al., Trans. Assoc. Am. Physicians 101:54, 1988; G. P. Rodgers et al., N. Engl. J. Med. 328:73-80, 1993), granulocyte/macrophage-colony stimulating factor (GM-CSF) (M. Giabbianeri et al., Blood 74:2657, 1989), and interleukin-3 (IL3) (A. R. Migliaccio et al., Blood 76:1150, 1990). Each of these factors were found to increase fetal globin synthesis in tissue culture cells.
Other agents shown to affect fetal globin expression include activin and inhibin. Inhibin, a disulfide linked hormone of two subunits, suppresses secretion of follicle-stimulating hormone from the pituitary gland. Activin, sometimes referred to as erythroid differentiating factor (EDF) or follicle-stimulating hormone releasing protein (FRP), is also a hormone and both of these macromolecules induced hemoglobin accumulation in cultured human erythrocytes (S. P. Perrine et al., Blood 74:114a, 1989). Recently, studies have shown that steel factor, a product of the mouse steel locus (D. M. Anderson et al., Cell 63:235-43, 1990), is also capable of influencing fetal globin synthesis in erythroid progenitors (B. A. Miller et al., Blood 79:1861-68, 1992).
Several studies have focused on the mechanism whereby butyric acid and other small organic molecules have been able to stimulate fetal globin expression (R. Oliva et al., Nuc. Acids Res. 18:2739, 1990). Experiments with cells in culture have indicated that butyric acid may act by increasing the level of histone acetylation by, possibly, decreasing the activity of one or more histone deacetylase. Resulting histone hyperacetylation may produce nucleosome unfolding and thereby increased gene expression. Other studies have indicated that hypo-methylation of the area of DNA around the .beta. gene complex correlates with increased .gamma. globin gene expression in thalassemic patients (S. Charache et al., Proc. Natl. Acad. Sci. USA 80:4842-46, 1983). Alternatively, butyric acid and other small molecules may function to increase specific gene expression by acting directly on agents which regulate transcription, the so-called transcription factors. These factors bind to sequence-specific sites along the genome at areas which control the expression of proximally located genes.
In contrast to the human alpha globin gene locus, the beta locus has been analyzed in great detail due, in part, to the identification of multiple mutations of beta globin genes in HPFH patients. The beta locus contains a large upstream sequence referred to as the locus control region (LCR), extending 8-16 kbp 5' of the epsilon gene. This sequence is divided into four DNase hypersensitive sites, HSS I-IV, that contain enhancer sequences, silencer sequences, transcription factor binding sites and other cis acting sequences (W. C. Forrester et al., Proc. Natl. Acad. Sci. USA 86:5439, 1989). Each of the genes of the beta globin cluster contains its own promoter which acts in concert with enhancer elements in the LCR. In fact, deletion of the LCR results in a thalassemic syndrome with little to no beta globin expression. These results indicate that the beta globin gene expressed may exert a competitive interaction over the LCR so that its enhancer effect is only available to a single gene at any given time of development (P. Fraser et al., Genes Dev. 7, 106-13, 1993).
A number of transcription factors have been identified in the beta locus which are thought to alter the level of beta globin gene expression. An enhancer element of the LCR has been shown to contain a pair of binding sites for nuclear factor E2 (NF-E2) which overlaps a tandem set of binding sites for transcription factor AP-1 (N. C. Andrews et al., Nature 362:722, 1993). NF-E2, a hematopoietic-specific basic leucine zipper protein, and AP-1 binding sites have been located on a variety of globin genetic elements (P. A. Ney et al., Genes Dev. 4:993, 1990). Recently, a conserved sequence (CS) located upstream of the AP-1/NF-E2 site has been proposed to augment enhancing activity (S. Safaya et al., Blood 84:3929-35, 1994).
Additional factors that bind to elements within the promoters of the beta globin cluster have been identified. The CAT box displacement protein (CDP) binds to the sequence CAAT, located about 50 bp upstream of many gene promoters. Another fairly ubiquitous transcription factor, SP1, binds to positions -140 and -202, and possible additional sites as well. TAFII110 has been shown to binds to the TATA box of many of the beta globin promoters (T. Hoey et al., Cell 72:247-60, 1993). Transcription factors GATA-1, binds to the transcription initiation site (GATA) and may be displaced by TFIID when forming an active initiation complex (M. C. Barton et al., Genes Dev. 7:1796-809, 1993). Another erythroid-specific factor, YY1, binds to at least 11 sites distributed throughout the globin regulatory region.
Recently, a factor has been identified that may be involved in the developmental regulation of hemoglobin expression. This factor, termed the stage selector protein (SSP), binds to a site located about 50-60 bps upstream of the gamma globin promoter referred to as the stage selector element (SSE) (S. M. Jane et al., EMBO J. 14:97-105, 1995). The SSE is also the site where a number of mutations have been found in HPFH syndrome patients. SSP has been purified from K562 cell nuclear extracts and its relatively fetal and erythroid specificity has been attributed to a heterodimeric partner protein of 40-45 kD termed CP2 which selectively allows assembly of the SSP complex on the SSE, and also on sites within the .epsilon. promoter, and subsequent interaction with RNA polymerase.